Multimeric peptide-MHC Class I (pMHCI) reagents enable the detection, characterization and purification of epitope-specific CD8+ T cells by flow cytometry and have greatly facilitated studies to advance our understanding of virus-specific CD8+ T cells. Studies that examine the development and evolution of virus- specific and cross-reactive CD8+ T cells are an important focus of this Program Project. Project I (Luzuriaga) will use the Epstein Barr virus (EBV) model of persistent viral infection to characterize the lineage relationship between effector and memory responses and the factors that influence the evolution of antigen-specific CD8+ T cell responses into the memory CD8+ T cell repertoire. Project 2 (Selin) will determine how cross-reactivity impacts CD8+ T cell selection and function, and influences disease outcome. The Tetramer Core will produce the large quantities of high-quality reagents crucial for both of these projects in a timely and cost effective manner. We utilize well-established techniques for the expression and purification of MHC I molecules, refolding with peptide epitopes, and conjugation with fluorochromes. We plan to expand our inventory to include additional pMHCI reagents that will enable a broader analysis of EBV-specific CD8+ T cell responses and will utilize newer fluorescent labels, such as QDots, to facilitate the simultaneous analyses of multiple EBV epitope-specific CD8+ T cell responses by multiparameter flow cytometry. Over the past funding period, we have developed reagents with altered CD8 binding capacity to better define differences in avidity of T cell receptor interactions with cognate antigen. We will also develop bi-specific heterodimers for characterizing cross-reactive CD8+ T cells.